A method of treating infection of sutures and prosthetic devices

ABSTRACT

An agent capable of inhibiting signalling mediated by a β 1  integrin cell surface receptor of leukocyte cells will treat a bacterial infection associated with a surface of a foreign body over and around which fibrin has been deposited, or a malignant tumor over and around which tenascin has been deposited. In addition, coating a foreign body with a fibrinolytic agent will prevent chronic bacterial infection associated with the surface of the foreign body. Furthermore, an agent capable of stimulating signalling mediated by a β 1  integrin cell surface receptor of leukocyte cells will treat chronic inflammation.

[0001] This application is a continuation-in-part application of U.S.Ser. No. 08/635,572, filed on Apr. 22, 1996, which is herebyincorporated by reference.

[0002] The invention disclosed herein was made with Government supportunder NIH Grant No. AI 20516 from the Department of Health and HumanServices. Accordingly, the U.S. Government has certain rights in thisinvention.

[0003] Throughout this application, various references are referred towithin parentheses. Disclosures of these publications in theirentireties are hereby incorporated by reference into this application tomore fully describe the state of the art to which this inventionpertains. Full bibliographic citation for these references may be foundat the end of this application, preceding the sequence listing and theclaims.

BACKGROUND OF THE INVENTION

[0004] Soluble or cell bound chemoattractants (1, 2), stimulatepolymorphonuclear leukocytes (PMN) to emigrate from the vasculature andmigrate toward sites of injury, infection, and inflammation. PMNsexpress unique plasma membrane receptors for many differentchemoattractants and cytokines [e.g., IL-8, leukotriene B4 (LTB4),formyl-methionyl-leucyl-phenylalanine (fMLP) and TNF-α] (3).Interactions between these receptors and soluble or surface-boundchemoattractants or cytokines signal PMNs to alter their expressionand/or activity of selectins and integrins (4, 5), and regulate PMNspatial orientation and movements. (6).

[0005] Tenascin, also referred to as cytotactin, hexabrachion, andglioma-mesenchymal extracellular matrix protein (41, 43, 44), forms adisulfide linked multimeric six-armed structure called a hexabrachion(43). Tenascin is expressed in many tissues during embryonicdevelopment, and is thought to play an important role in the developmentof muscles and tendons, mammary glands, hair follicles, teeth, kidney,bone and cartilage (43, 50, 51). In adults, tenascin is expressed inT-cell dependent regions of lymphoid tissues (40), in areas of cellularinjury, and in malignant, but not benign tumors (41, 43). In areas ofinjury, tenascin is present in granulation tissue (41, 55), inassociation with proliferating and migrating epidermal cells (42, 49),and in arteries whose endothelial cells have been damaged (57). Inmalignant neoplasms, tenascin is produced by the tumor cells (63) anddeposited in the stroma of gliomas, mammary carcinomas, colon cancers,Wilm's tumor, basal cell carcinomas, melanomas, and squamous cellcarcinomas (41, 64).

[0006] There is little information regarding the physiological orpatho-physiological role(s) of tenascin at sites of tissue injury,malignancy or atherosclerotic lesions. In extracellular matrices,tenascin promotes the adhesion of endothelial cells and bone marrowcells (38, 48). Tenascin also blocks the attachment of several othercell types to fibronectin-coated surfaces in vitro (47, 52), and themigration of neural crest cells (20, 43, 62).

SUMMARY OF THE INVENTION

[0007] The present invention provides a method of treating an infectioncaused by bacterial cells located on a surface of a foreign body overand around which fibrin has been deposited, the foreign body beingpresent in a subject, which comprises administering to the subject anagent capable of inhibiting signalling mediated by a β₁ integrin cellsurface receptor of leukocyte cells in an amount effective to enhancethe migration of leukocyte cells into or through the fibrin so as topermit the leukocyte cells to reach and kill the bacterial cells andthereby treat the infection.

[0008] The present invention also provides a method of preventing achronic infection from occurring due to the presence of bacterial cellson a surface of a foreign body in a subject, which comprises coating theforeign body before placing it in the subject with a fibrinolytic agentcapable of preventing the accumulation of fibrin on the surface of theforeign body so as to permit leukocyte cells to reach and kill anybacterial cells present on the surface of the foreign body and therebyprevent the chronic infection.

[0009] The present invention further provides a method of treating amalignant tumor comprising of malignant tumor cells over and aroundwhich tenascin has been deposited, the malignant tumor being present ina subject, which comprises administering to the subject an agent capableof inhibiting signalling mediated by a β₁ integrin cell surface receptorof leukocyte cells in an amount effective to enhance the migration ofleukocyte cells through the tenascin so as to permit the leukocyte cellsto reach and kill the malignant tumor cells and thereby treat themalignant tumor.

[0010] The present invention also provides a method of treating achronic inflammation in a subject caused by an increase in the number ofleukocyte cells present at the site of the chronic inflammation whichcomprises administering to the subject an agent capable of stimulatingsignalling mediated by a β₁ integrin cell surface receptor of leukocytecells in an amount effective to inhibit the migration of leukocyte cellstoward the site of the chronic inflammation so as to reduce the numberof leukocyte cells present at the site and thereby treat the chronicinflammation.

BRIEF DESCRIPTION OF THE FIGURES

[0011]FIGS. 1A and 1B show that IL-8 and LTB4 promote PMN migrationthrough fibrin gels and plasma clots. Fibrin gels (A) or plasma clots(B) were formed on top of filters with 8 μm pores in tissue cultureinserts as described under Materials and Methods. 10⁶ PMNs were added tothe upper chamber and the indicated chemoattractants or cytokines wereadded to either the lower or upper chamber as indicated. The preparationthen was incubated at 37° C. for 6 hrs, at which time the number ofcells in the lower chamber was determined using a Coulter counter.Concentrations of chemoattractants/cytokines used were: 0.75×10⁻⁷ M forIL-8, 1.0×10⁻⁷ M for LTB4, 0.5×10⁻⁷ M for TNF and 1.0×10⁻⁷ M for fMLP.Fewer than 1500 PMNs migrated through fibrin gels in the absence of anystimulator.

[0012]FIGS. 2A and 2B show PMN migration through fibrin gels in responseto varying concentrations of chemoattractants [fMLP, TNF (A); LTB4, IL-8(B)]. Fibrin gels were formed on filters of cell culture inserts asdescribed in Experimental Procedures and chemoattractants or cytokinesat the indicated concentrations were added to the lower chamber. 10⁶PMNs were added to the upper chamber and the preparation was incubatedat 37° C. for 6 hrs, at which time the number of cells in the lowerchamber was counted as described in FIGS. 1A and 1B.

[0013]FIGS. 3A and 3B show that an IL-8 or LTB4 gradient is required forPMN migration through fibrin gels. Fibrin gels were formed in cellculture inserts as described in Experimental Procedures. IL-8 (A) orLTB4 (B) were added to the lower compartment at a fixed concentrationand to the upper compartment at varying concentrations as indicated. 10⁶PMNs were added to the upper chamber and the preparation was incubatedat 37° C. for 6 hrs, at which time the number of cells in the lowerchamber was counted as described in FIGS. 1A and 1B.

[0014]FIG. 4 shows a time course of PMN migration through fibrin gels.Fibrin gels were formed in cell culture inserts as described underExperimental Procedures. IL-8 (0.75×10⁻⁷ M) or LTB4 (10⁻⁷ M) were addedto the lower chamber. The number of PMNs that migrated through a fibringel was determined at the indicated times as described in FIGS. 1A and1B.

[0015]FIGS. 5A, 5B, 5C, 5D, 5E and 5F show a confocal microscopicanalysis of PMN migration through fibrin gels. Fibrin gels were formedas described under Experimental Procedures and chemoattractants orcytokines at the indicated concentrations were added to the lowerchamber. 10⁶ PMNs, prelabeled with calcein as described underExperimental Procedures, were added to the upper chamber at 37° C. Atthe indicated times, the filters were removed from the inserts, washedand viewed by confocal microscopy as described under ExperimentalProcedures. All samples were viewed en-face, the images were rotated 90°C. The surface of the gel is marked with a bar. (A and B), fMLP (10⁻⁷M); (C and D), TNF (0.5×10⁻⁷ M); (E and F), IL-8 (0.75×10⁻⁷ M)

[0016]FIG. 6 shows that degradation of ¹²⁵I-fibrin by PMNs does notaccount for the increased of IL-8 of LTB4 stimulated PMNs. Cell cultureinserts containing 125I-labeled fibrin gels were prepared as described.10⁶ PMNs were added to the insert and the indicatedchemoattractants/cytokine were added to the lower compartment asdescribed in FIGS. 1A and 1B. The concentrations of chemoattractantsused are the same as described in FIGS. 5A, 5B, 5C, 5D, 5E, and 5F.After a 6 hr incubation at 37°, media from both the upper and lowercompartments were collected and the assayed for the amount ofradioactivity in TCA-soluble and insoluble fractions as described inMaterials and Methods. This figure is representative of experimentsrepeated three times with similar results. The data in this figure arethe average of values from duplicate samples.

[0017]FIGS. 7A and 7B show PMN migration through filters coated withdifferent types of matrix proteins. PMN migration through filters coatedwith gels formed with reconstituted basement membrane (Matrigel), orcollagen IV (A), or with filters coated with fibrinogen, or fibronectin(B), was measured in response to TNF (5×10⁻⁷ M), fMLP (10⁻⁷ M), IL-8(0.75×10⁻⁷ M) or LTB4 (10⁻⁷ M) Migration was essentially complete within2 hr. The last column of FIG. 5A reports migration of TNF-stimulated PMNthrough a collagen IV gel impregnated with fibrin.

[0018]FIG. 8 shows “Closeness” of PMN apposition to fibrin matrices.PMNs stimulated with the indicated chemoattractants were allowed toadhere for 15 min to glass surfaces coated with fibrin as describedunder Experimental Procedures. PMNs forming close zones of adhesion aredefined as those that exclude the entry of Rh-PEG into the area ofadhesion between the cell and the underlying matrix as assayed byfluorescence microscopy. The concentrations ofchemoattractants/cytokines used were the same as in FIG. 6. Less than20% of unstimulated PMNs that adhered to the fibrin formed close zonesof apposition.

[0019]FIGS. 9A and 9B show the effect of combinations ofchemoattractants/cytokines in promoting PMN migration through fibringels. PMN migration through fibrin gels was measured in response to TNF(A) or fMLP (B) added in combination to the bottom compartment, at theconcentrations indicated. The concentrations of IL-8 and LTB4 used werethe same as in FIGS. 5A, 5B, 5C, 5D, 5E, and 5F. PMN migration into thelower compartment was measured as described in FIGS. 1A and 1B after a 6hr incubation at 37° C.

[0020]FIGS. 10A and 10B show that fMLP promotes PMN migration throughfibrin gels when these cells are treated with antibodies directedagainst β₁ integrins (A) or incubated in medium containing syntheticpeptide (B). Fibrin gels were formed on filters of cell culture insertsas described in Experimental Peocedures and fMLP (10⁻⁷M) or LTB4 (10⁻⁷M) was added to the medium in the lower chamber. FIG. 10A—10⁶ PMNswere pre-incubated in medium containing antibodies against rat or humanβ₁ integrins, or antibodies against the α₅ subunit of human α₅β₁integrin, or against the human β₂ integrin complement receptor 3 (CR3),or against the human β₂ integrin p150/95, or against human β₃ integrinsfor 15 min at 4° C. PMNs were placed in the upper chamber of cellculture inserts in the same medium and the inserts were incubated for 6hrs at 37° C., at which time the number of cells in the lower chamberwas determined using a Coulter Counter. FIG. 10B—10⁶ PMNs were placed inmedium containing the synthetic peptide GRGDSP (SEQUENCE ID NO. 1) (1mg/ml) or the peptide GRGESP (SEQUENCE ID NO. 2) (1 mg/ml). The mixturewas added to the upper chamber of cell culture inserts and the insertswere incubated for 6 hrs at 37° C., at which time the number of cells inthe lower chamber was counted as in FIG. 10A.

[0021]FIGS. 11A and 11B show that tenascin inhibits the chemotaxis ofmononuclear phagocytes through Matrigel-coated filters. Cell insertscoated with either Matrigel or Matrigel and chick tenascin were preparedas described under Experimental Procedures. 10⁶ freshly harvestedmonocytes (A), or monocytes cultured for 24 h (B), were then added tothe upper compartment and the indicated chemoattractant [TNF (5×10⁻⁷ M),LTB4 (10⁻⁷ M) or fMLP (10⁻⁷ M)] was added to the lower compartment. Theinserts were incubated for 24 h at 37° C. and the cells that migratedinto the lower compartment were counted using a Coulter Counter.

[0022]FIG. 12 shows that chick tenascin inhibits the chemotaxis of PMNsthrough Matrigel-coated filters. Cell inserts coated with eitherMatrigel or Matrigel and chick tenascin were prepared as described underExperimental Procedures. 10⁶ freshly isolated PMNs were added to theupper compartment and the indicated chemoattractant [TNF (5×10⁻⁷), LTB4(10⁻⁷ M), or fMLP (10⁻⁷ M)], was added to the lower compartment. Theinserts were incubated for 2-4 h at 37° C. and the cells that migratedinto the lower compartment were counted using a Coulter Counter as inFIGS. 11A and 11B.

[0023]FIGS. 13A and 13B show that human tenascin inhibits the chemotaxisof monocytes or PMNs through Matrigel coated filters. Cell insertscoated with either Matrigel or Matrigel and human tenascin were preparedas described under Experimental procedures. 5×10⁵ freshly isolatedmonocytes, or 10⁶ PMNs were added to the upper compartment and theindicated chemoattractant [fMLP (10⁻⁷ M) or LTB4 (10⁻⁷ M)] was added tothe lower compartment. Migration of monocytes (A) was allowed to proceedfor 24 h at 37° C. Migration of PMNs (B) was allowed to proceed for 6 hat 37° C. and the cells that migrated into the lower compartment werecounted using a Coulter Counter as in FIGS. 11A and 11B.

[0024]FIG. 14 shows that chick tenascin inhibits the migration ofcultured monocytes through Matrigel-coated filters in a dose dependentfashion. Cell inserts were coated with Matrigel and then incubated withPBS containing the indicated concentrations of tenascin. The filterswere washed with PBS. 2×10⁵ cultured monocytes were added to the uppercompartment. TNF (5×10⁻⁷M) was added to the lower compartment and thecells were allowed to migrate for 24 h at 37° C. This experiment isrepresentative of 3 experiments. Fewer than 1500 cultured monocytesmigrated in the absence of any chemoattractant.

[0025]FIG. 15 shows binding of radiolabeled tenascin to Matrigel-coatedfilters. ¹²⁵I-labeled chick tenascin was prepared as described underExperimental Procedures and mixed with unlabeled tenascin at a proteinratio of 1:100. This mixture was added to Matrigel-coated inserts in theamounts indicated and the inserts were incubated at 37° C. for 4 h,conditions identical to those used for the migration experimentsdescribed in FIGS. 11A and 11B. The filters then were washed, cut fromthe inserts, and counted in a LKB γ-counter.

[0026]FIG. 16 shows the effects of tenascin on the migration of culturedmonocytes through filters coated with collagen I. Cell inserts werecoated with 40 μg of collagen I. PBS containing 5 μg of chick tenascinwas added to some of the inserts for 4 h as described under ExperimentalProcedures. 5×10⁵ cultured monocytes were added to the upper compartmentof the inserts and allowed to migrate for 24 h in the absence of achemoattractant or in response to TNF (5×10⁻⁷ M) or LTB4 (10⁻⁷ M) in thelower compartment. The cells that migrated into the lower compartmentwere counted as described in FIGS. 11A and 11B.

[0027]FIGS. 17A and 17B show that F(ab)′₂ fragments of anti-tenascinantibodies or anti-β₁ monoclonal antibodies promote monocyte and PMNchemotaxis through Matrigel/tenascin coated filters. 10⁶ freshlyisolated monocytes (A) or PMNs (B) were used as described in FIGS. 11A,11B and 12. Where indicated the cells were pre-incubated for 30 min at4° C. with various antibodies [2 μg/ml] before adding them to the uppercompartment of the chemotaxis chambers. The antibodies used wereanti-tenascin (46), P4C10 (anti-β₁), and LeuM5 (7) (anti-α_(x)β₂). Thecells that migrated into the lower compartment at 24 h (A) and at 4 h(B) were determined as described in FIGS. 11A and 11B.

DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention provides a method of treating an infectioncaused by bacterial cells located on a surface of a foreign body overand around which fibrin has been deposited, the foreign body beingpresent in a subject, which comprises administering to the subject anagent capable of inhibiting signalling mediated by a β₁ integrin cellsurface receptor of leukocyte cells in an amount effective to enhancethe migration of leukocyte cells into or through the fibrin so as topermit the leukocyte cells to reach and kill the bacterial cells andthereby treat the infection.

[0029] In one embodiment of the invention, the foreign body is aprosthetic device, a catheter, or a suture.

[0030] In another embodiment of the invention, the subject is a mammalsuch as a human.

[0031] In yet another embodiment of the invention, the leukocyte cellsare polymorphonuclear leukocyte cells such as neutrophils, basophils,and eosinophils.

[0032] In another embodiment of the invention, the leukocyte cells aremonocytes or macrophages.

[0033] In yet another embodiment of the invention, the agent is apeptide such as a peptide containing a β₁ integrin-binding domain,specifically one comprising GRGDSP. The agent can also be apeptidomimetic compound.

[0034] In another embodiment of the invention, the agent is an antibodyor a fragment thereof that specifically binds to the β₁ integrin cellsurface receptor of leukocyte cells.

[0035] The present invention also provides a method of preventing achronic infection from occurring due to the presence of bacterial cellson a surface of a foreign body in a subject, which comprises coating theforeign body before placing it in the subject with a fibrinolytic agentcapable of preventing the accumulation of fibrin on the surface of theforeign body so as to permit leukocyte cells to reach and kill anybacterial cells present on the surface of the foreign body and therebyprevent the chronic infection.

[0036] In one embodiment of the invention, the foreign body is aprosthetic device, a catheter, or a suture.

[0037] In another embodiment of the invention, the subject is a mammalsuch as a human.

[0038] In yet another embodiment of the invention, the fibrinolyticagent is plasminogen activator such as urokinase, streptokinase, ortissue plasminogen activator.

[0039] The present invention further provides a method of treating amalignant tumor comprising of malignant tumor cells over and aroundwhich tenascin has been deposited, the malignant tumor being present ina subject, which comprises administering to the subject an agent capableof inhibiting signalling mediated by a β₁ integrin cell surface receptorof leukocyte cells in an amount effective to enhance the migration ofleukocyte cells through the tenascin so as to permit the leukocyte cellsto reach and kill the malignant tumor cells and thereby treat themalignant tumor.

[0040] In one embodiment of the invention, the subject is a mammal suchas human.

[0041] In another embodiment of the invention, the leukocyte cells arepolymorphonuclear leukocyte cells such as neutrophils, basophils, andeosinophils.

[0042] In yet another embodiment of the invention, the leukocyte cellsare monocytes or macrophages.

[0043] In another embodiment of the invention, the leukocyte cells arelymphocyte cells such as NK cells and killer cells.

[0044] In yet another embodiment of the invention, the agent is apeptide such as a peptide containing a β₁ integrin-binding domain,specifically one comprising GRGDSP. The agent can also be apeptidomimetic compound.

[0045] In another embodiment of the invention, the agent is an antibodyor a fragment thereof that specifically binds to the β₁ integrin cellsurface receptor of leukocyte cells.

[0046] The present invention also provides a method of treating achronic inflammation in a subject caused by an increase in the number ofleukocyte cells present at the site of the chronic inflammation whichcomprises administering to the subject an agent capable of stimulatingsignalling mediated by a β₁ integrin cell surface receptor of leukocytecells in an amount effective to inhibit the migration of leukocyte cellstoward the site of the chronic inflammation so as to reduce the numberof leukocyte cells present at the site and thereby treat the chronicinflammation.

[0047] In one embodiment of the invention, the subject is a mammal suchas human.

[0048] In another embodiment of the invention, the leukocyte cells arepolymorphonuclear leukocyte cells such as neutrophils, basophils, andeosinophils.

[0049] In yet another embodiment of the invention, the leukocyte cellsare monocytes or macrophages.

[0050] In yet another embodiment of the invention, the agent is apeptide such as a peptide containing a β₁ integrin-binding domain,specifically one comprising GRGDSP. The agent can also be apeptidomimetic compound.

[0051] This invention will be better understood from the ExperimentalDetails which follow. However, one skilled in the art will readilyappreciate that the specific methods and results discussed are merelyillustrative of the invention as described more fully in the claimswhich follow thereafter.

EXPERIMENTAL DETAILS Example 1

[0052] The evolution of many chemically distinct chemoattractants andreceptors suggests that in addition to promoting adhesion and directingPMN locomotion these molecules might regulate the strength of PMNadhesion to specific matrix proteins. The findings that TNF stimulatesPMNs to adhere to fibrinogen-coated surfaces via CD11c/CD18 (7, 8),while phorbol dibutyrate stimulates PMNs to adhere to these surfaces viaCD11b/CD18 (8, 9), prompted the examination of the effects of differentchemoattractants on PMN migration through three dimensional matricescomposed of fibrin, collagen IV or Matrigel, and through gels formed bythrombin treatment of cell-free plasma.

EXPERIMENTAL PROCEDURES Reagents

[0053] Human monocyte recombinant IL-8 (Ser-IL-8)₇₂ and TNF-α were fromUpstate Biotechnology Incorporated (Lake Placid, N.Y.). LTB4, fMLP andFicoll-Hypaque were from Sigma (St. Louis, Mo.). Rhodamine conjugatedpolyethylene glycol was prepared as described (10).

Preparation of Boyden-Type Chemotaxis Chambers

[0054] Becton-Dickinson cell culture inserts (pore sizes 3 or 8 μm:Franklin Lakes, N.J.) were overlaid with the following proteins:

[0055] Fibrin gels: 1 Unit of thrombin (a gift from Dr. John Fenton,Albany Medical College, Albany, N.Y.), in 5 μl of PBS was added first toeach insert. 0.1 ml phosphate buffered saline supplemented with Ca²⁺ andMg²⁺ (PBS) containing 100 μg commercial grade fibrinogen (Calbiochem.Inc., San Diego, Calif.) or purified fibrinogen [a gift from Dr. JefferyWeitz, (MacMaster University, Hamilton, On.)] was then placed into each8.2 mm diameter insert on top of the thrombin. The mixture was incubatedat 37° C. for 5 min to allow fibrin gel formation (determined by visualinspection). 1 Unit PPACK (Calbiochem Inc., San Diego, Calif.; 10⁻⁵ Mfinal concentration), in 100 μl medium was added to each insert toinhibit thrombin, and gels were washed with 250 μl PBS to removeinactivated thrombin. The fibrin gels formed were about 1 mm thick asmeasured under a dissecting microscope.

[0056] Collagen type IV and Matrigel matrices: 0.1 ml PBS containing 100μg human placental collagen IV (Fluka Chemical Corp., Ronkonkoma, N.Y.),or 80 μg of reconstituted basement membrane proteins (Matrigel,Collaborative Research, Bedford, Mass.), was placed into each insert andallowed to gel at room temperature for 24 hrs.

[0057] Clotted Plasma: Whole blood was collected and the cellularcomponents were removed by centrifugation. The resulting plasma wasmixed with an equal volume of PBS, and 100 μl of this mixture was placedinto each insert containing thrombin and allowed to clot as describedabove. One unit of PPACK in 100 μl PBS then was added and the insertswere washed with 250 μl of PBS.

[0058] Fibrinogen or fibronectin: 0.1 ml of PBS containing 100 μg/mlfibrinogen or fibronectin (New York Blood Center, New York, N.Y.), wasplaced into each insert (pore size 3 μm). Inserts were incubated at 37°C. for 60 min and washed with 250 μl of PBS. Filters coated withfibrinogen or fibronectin were diffusely fluorescent as visualized byepifluorescent microscopy when incubated with the corresponding antibody[fluorescein-labeled anti-fibrinogen, or anti-fibronectin monoclonalantibodies (Cappel, Malvern, Pa.)], while uncoated filters, or filtersincubated with fluorescein-labeled antibody of the opposite specificity,were not.

PMN Migration

[0059] PMNs were prepared from fresh heparinized blood from healthyadult volunteers by sedimentation on Ficoll-Hypaque gradients.Contaminating red blood cells were removed by hypotonic lysis, asdescribed (7). The purity of PMN isolated by this method is >95% asdetermined by Wright-Giemsa staining (7). 10⁶ PMNs in 250 μl of PBSsupplemented with 5.5 mM glucose and 0.1% human serum albumin(PBSG-HSA), were placed in the upper compartment of each insert andincubated for 0-6 hrs at 37° C. in a humidified atmosphere containing95% air/5% CO₂. At the times and concentrations specified,chemoattractants/cytokines were added to the top or bottom compartmentin 250 μl of PBSG-HSA. At the end of the incubation, the chambers wereshaken to dislodge PMNs from the lower surface of the inserts. Themedium in each lower compartment was collected and its content of PMNswas determined using either a Coulter Counter or a hemocytometer. Bothmethods gave similar results. Counts are expressed as the average numberof PMNs that migrated into the lower compartment. Unless otherwiseindicated, all values reported are the average of six data points fromat least 3 independent experiments.

Confocal Microscopy

[0060] PMNs were suspended in medium containing 10 μMcalcein/acetoxymethyl ester (Molecular Probes, Eugene, Oreg.), 0.02%(w/v) pluronic F-127 (Molecular Probes, Eugene, Oreg.), 2% heatinactivated calf serum (HyClone, Logan, Utah), and 0.2% DMSO, and mixedgently for 40 min at room temperature. Cells loaded with dye under theseconditions exhibited no changes in motility (Mandeville and Maxfield,unpublished observations). The calcein-loaded cells were rinsed inPBSG-HSA and added to inserts containing fibrin gels in the presence orabsence of TNF, fMLP, LTB4 or IL-8. Following incubation with PMNs,fibrin-coated filters were gently cut from their inserts using a razorblade, transferred to a glass slide, immersed in PBSG-HSA and coveredwith a glass coverslip. Migration of calcein-loaded PMNs through fibrinwas analyzed using a Dialux 20× microscope (Leitz) fitted with a K2 Bioconfocal scanning optical attachment using a Nipkow spinning disk. Themicroscope was equipped with an image intensifier, charge coupled devicecamera and video frame averager. The surface of the fibrin gel wasidentified using reflection interference contrast microscopy. Cells wereimaged with a Plan-neofluor 25× fluorescence objective (NA=0.8) usingfluorescein optics (490 nm excitation, 525 emission) and a spinning diskwith pinhole apertures. Serial confocal optical sections were acquiredat 1 μm intervals, digitized using the VolCon program (a PC-based imageprocessing package, Indec Systems, Capitola, Calif.,). Three dimensionalimages were volume rendered using Microvoxel software (Index Systems),after passing data through a 3×3×3 Gaussian convolution filter. Eachexperiment was repeated at least twice using duplicate samples.

PMN Adhesion to Fibrin-Coated Surfaces

[0061] Fibrin coated Terasaki tissue culture plates were prepared asdescribed (10). 5 μl of PBSG-HSA, containing PMNs (10⁶/ml) and theindicated chemoattractant, was added to each well of the plate. Plateswere incubated at 4° for 30 min to allow PMNs to settle to the bottom ofthe wells and warmed to 37° for 15 min to allow PMNs to adhere.Non-adherent cells were removed as described (7), and 2.5%glutaraldehyde in PBS added to fix the adherent PMNs. PMNs adherent toeach well were enumerated using a phase-contrast microscope. Valuesreported are the mean number of PMNs adherent to six wells from arepresentative experiment (n=3).

Exclusion of Rhodamine-Labeled Polyethylene Glycol from Zones ofAdhesion of PMNs to Protein-Coated Surfaces

[0062] 10 kDa rhodamine labeled polyethylene glycol (Rh-PEG), preparedand used as described previously (10), does not bind to untreated glass,tissue culture plastic, or to cell membranes. Rh-PEG binds avidly toprotein-coated surfaces, and can be detected easily by its fluorescence.Individual wells on glass microslides (Carlson Scientific, Peotone,Ill.) were coated with either fibrin, Matrigel or collagen IV in amanner similar to that for coating cell culture inserts except that 20μl of the various solutions were used per well. 20 μl of PMNs (10⁶cells/ml in PBSG-HSA) were added to each well and PMNs were allowed toadhere for 15 mins at 37°. The cells were washed in PBS, fixed with 3.7%paraformaldehyde in PBS for 10 min, washed again with PBS, and furtherincubated with 10 kDa Rh-PEG at room temperature for 60 min. Thepreparation then was washed with PBS and immediately observed by phaseand fluorescent microscopy at 400× magnification. Average values fromthree different experiments are reported as the percentage of PMNs thatexcluded Rh-PEG from zones of adherence between the cells and theunderlying matrix.

Degradation of ¹²⁵I-Labeled Fibrin Gels

[0063] 1.0 ml PBS containing 1 mg human fibrinogen, 1 mCi of Na¹²⁵I(NEN, Boston, Mass.), and 1 Iodobead (Pierce, Rockford, Ill.) wasincubated for 15 mins on ice. ¹²⁵I-fibrinogen was separated from ¹²⁵I bygel filtration over a Speedy Desalting Column (Pierce). >97% of the ¹²⁵Irecovered in the fibrinogen-containing fractions was precipitable with20% trichloracetic acid. 5 μl PBS containing 1 Unit of thrombin,followed by 0.1 ml PBS containing 10⁶ cpm of ¹²⁵I-fibrinogen (˜10 μg)and 100 μg unlabeled fibrinogen were added to each insert, as describedabove. The resulting ¹²⁵I-labeled fibrin gels were treated with PPACK,washed, and incubated with 10⁶ PMNs as described in the text. At varioustimes after PMN addition the medium was removed from the upper and lowercompartments, and added to 0.1 ml of PBS containing 10 mg/ml bovineserum albumin (BSA). Ice-cold trichloracetic acid was added to a finalconcentration of 20% and samples were centrifuged to sediment acidinsoluble materials. TCA soluble and insoluble materials were separatedby centrifugation and ¹²⁵I in each fraction was determined using an LKBminigamma counter.

Results IL-8 and LTB4, but Not TNF or fMLP, Promote the Migration ofPMNs through Fibrin Gels

[0064] PMNs were placed into the upper compartment of inserts containingfibrin gels. IL-8, LTB4, fMLP, or TNF was placed in the medium in thelower compartment and the chambers were incubated at 37° C. for 6 hrs.IL-8 or LTB4 stimulated 12-25% of PMNs to migrate through the fibringels and into the lower compartment. In the absence of a chemoattractantor in response to various concentrations of TNF (10⁻⁹-5×10⁻⁶ M) or fMLP(10⁻¹⁰-10⁻⁶ M), fewer than 0.3% of the PMNs migrated through fibrin gelsinto the lower compartments (FIGS. 1A and 2). Moreover, PMNs did notmigrate through fibrin in response to the addition of 2-10%zymosan-activated human plasma (C5a) in the lower compartment.

[0065] PMNs stimulated by IL-8 or LTB4, but not by TNF or fMLP, migratedthrough fibrin gels formed by thrombin treatment of commercial-gradefibrinogen (FIG. 1A) or of purified fibrinogen, or through plasma gelsformed by thrombin treatment of human plasma (FIG. 1B). Moreover, thepresence of 20% human serum in the medium in both upper and lowercompartments did not alter PMN migration through fibrin gels in responseto IL-8, nor did the presence of serum promote PMN migration throughfibrin gels in response to TNF or fMLP. That PMNs migrate through fibringels in the presence of human serum, and through gels formed from wholehuman plasma, indicates that IL-8 promotes PMN migration through fibringels containing the complex mixture of plasma proteins found underphysiological conditions.

[0066] The percent of PMNs that migrated through fibrin gels varied withthe concentration of IL-8 or LTB4 placed in the bottom compartment (FIG.2B). Maximal PMN migration occurred with 0.7×10⁻⁷ M IL-8 or 0.2×10⁻⁷ MLTB4 (FIG. 2B). PMN migration decreased dramatically when IL-8 was usedat concentrations >10⁻⁷ M, consistent with the report of Smith et. al.(11) that high concentrations of IL-8 desensitize PMNs. In contrast,there was no indication of PMN desensitization in response tosupra-optimal concentrations of LTB4 (FIG. 2B).

[0067] To determine whether IL-8 and LTB4 promote PMN migration throughfibrin gels by stimulating chemotaxis or chemokinesis we performed acheckerboard-type analysis (12). Few PMNs migrated through fibrin gelswhen IL-8 or LTB4 was placed in the upper compartment, or when the upperand lower compartments contained equal concentrations of IL-8 or LTB4(FIGS. 1A and 1B). As the difference in IL-8 or LTB4 concentrationsbetween the upper and lower compartments decreased, the number of PMNsthat migrated through the fibrin gels also decreased (FIGS. 3A and 3B).These results indicate that PMN migration through fibrin gels inresponse to IL-8 or LTB4 reflects chemotaxis, not chemokinesis.

[0068] Between 25-50% more PMNs migrated through fibrin in response toLTB4 than to IL-8. It is unlikely that this difference reflects theresponse of different PMN subpopulations to LTB4 vs IL-8 since the samepercentage of PMNs traversed fibrin gels in response to optimalconcentrations of both LTB4 and IL-8 in the lower compartment as to LTB4alone. Other investigators have reported that only 20-50% of PMNsmigrate through filters (13), natural matrices, and cellular barriers(14), when stimulated by these chemoattractants. Since virtually allPMNs orient and crawl on surfaces when exposed to the chemoattractants(3), it is evident that all PMNs responded to them. The reason(s) whyonly a fraction of PMNs migrate through artificial or natural barriersin response to chemoattractants is unknown.

[0069] PMNs migrated through fibrin gels more rapidly in response to anoptimal concentration of LTB4 than to an optimal concentration of IL-8(FIG. 4). Ten percent of LTB4-stimulated PMNs migrated through fibringels within 2 hrs while fewer than 0.5% of IL-8-stimulated PMNs migratedthrough these gels in this time period (FIG. 4). By 6 hrs, maximalnumbers of PMNs had migrated through fibrin gels in response to eitherIL-8 or LTB4.

[0070] To visualize the interactions of chemoattractant-stimulated PMNswith fibrin gels, PMNs prelabeled with calcein (15), were added to theupper compartment of inserts containing fibrin gels. Chemoattractantswere added to the medium in the lower compartment, the chambers wereincubated at 37° C., and at the times indicated the fibrin-coatedfilters were removed and examined by confocal microscopy. After a 1 or 4hr incubation with fMLP or TNF, almost all the cells remained on thegel's surface; fewer than 5% of TNF- or fMLP-stimulated PMNs penetrateda short distance into the fibrin gels; (FIGS. 5A, 5B, 5C, 5D, 5E, and5F). In contrast, greater than 80% of IL-8-stimulated PMNs migrateddeeply into the fibrin gels after a 4 hr incubation (FIGS. 5A, 5B, 5C,5D, 5E, and 5F). Greater than 80% of LTB4-stimulated PMNs began tomigrate into the fibrin gel after 1 hr, while few IL-8 stimulated PMNspenetrated the fibrin at this time. These results show that TNF and fMLPdo not promote PMN invasion of fibrin, and that LTB4 stimulates PMNs toenter fibrin gels more rapidly than IL-8. The latter finding isconsistent with the more rapid transit of fibrin gels by LTB4- thanIL-8-stimulated PMNs described in FIG. 4.

[0071] To further examine whether proteolysis of fibrin accounted forthe selective ability of IL-8- or LTB4-stimulated PMNs to traverse thesegels, the release of ¹²⁵I-labeled products was measured from ¹²⁵I-fibrinincubated with PMNs for 6 hrs at 37° C. in the presence or absence ofeach of these chemoattractants. The rare and extent of release of¹²⁵I-labeled acid soluble and acid precipitable products was similar forall four chemoattractants (FIG. 6). Even in the presence of 20% serum,chemoattractant-stimulated PMNs released no more ¹²⁵I-labeled productsthan unstimulated PMNs. These results suggest that fibrin degradationdoes not account for the selective ability of LTB4- and IL-8-stimulatedPMNs to traverse fibrin gels.

LTB4, IL-8, TNF and fMLP Promote PMN Migration through Gels Formed ofMatrigel and Collagen IV

[0072] To confirm that the inability of TNF-, fMLP-, orzymosan-activated human plasma-stimulated PMNs to migrate through fibringels reflected an effect of the interaction between the fibrin matrixand chemoattractant-stimulated PMNs, and not a general effect of anythree dimensional matrix on PMNs stimulated with these chemoattractants,the ability of TNF, zymosan activated human plasma and fMLP to promotePMN migration through gels composed of basement membrane proteins(Matrigel) or collagen IV was examined (FIGS. 7A and 7B). TNF, fMLP,IL-8, zymosan-activated human plasma, or LTB4 added to the bottomchamber stimulated PMN migration through these gels (FIG. 7A). Todetermine whether fibrin affected PMN migration through collagenmatrices, inserts coated with collagen IV gels were incubated withfibrinogen and thrombin to form fibrin sandwich on top of the collagengels, and washed with PPACK-containing buffer. PMNs were added to theupper compartment and TNF to the lower compartment. The presence offibrin prevented PMN migration through the collagen gels in response toTNF by about 75% (FIG. 7A). These results confirm that the effect offibrin is selective and that it affects PMN migration in response to aspecific subset of chemoattractants.

[0073] To determine whether protein monolayers had the same effects onPMN migration as gels, inserts were coated with fibrinogen orfibronectin. The adsorption of these proteins to the filters that formthe floor of the inserts was confirmed by immunofluorescence microscopy,as described in Experimental Procedures. fMLP, TNF, IL-8 and LTB4 allpromoted PMN migration through filters to which fibrinogen orfibronectin had been adsorbed (FIG. 7B).

PMNs Adhere More Closely to Fibrin in Response to fMLP or TNF than toLTB4 or IL-8

[0074] Is there a relationship between the ability of a chemoattractantto stimulate PMN migration through fibrin and its ability to promoteclose apposition of PMNs to fibrin? PMNs were incubated on fibrin-coatedsurfaces in the presence or absence of a chemoattractant for 15 min at37° C. As expected, TNF, fMLP, LTB4 and IL-8 were equally effective instimulating PMN adherence to fibrin (>200 chemoattractant-stimulatedPMNs vs ˜10 unstimulated PMNs adhered per mm²). The closeness of PMNadhesion to fibrin was evaluated by the ability of 10 kDa Rh-PEG (8,10), to penetrate into the zones of adhesion betweenchemoattractant-stimulated PMNs and fibrin. By this measure 70-80% ofadherent TNF- or fMLP-stimulated PMNs excluded Rh-PEG from their zonesof contact with the fibrin (FIG. 8). In contrast, only about 15% ofadherent LTB4- or IL-8-stimulated PMNs formed adhesive zones thatexcluded Rh-PEG (FIG. 8). Furthermore, the adhesive zones formed by this15% of IL-8-stimulated PMNs were at least 50% smaller in area than thoseformed by TNF- or fMLP-stimulated PMNs as judged by the area from whichRh-PEG was excluded.

[0075] Previous studies (10) showed that the exclusion offluorescein-conjugated F(ab)₂ anti-fibrinogen from zones of contactbetween ADP-stimulated platelets and fibrinogen-coated surfaces is auseful measure of the closeness of apposition between platelet membranesand the substrate. Therefore, the exclusion of fluorescein-conjugatedF(ab)₂ anti-fibrin from zones of contact between LTB4- orIL-8-stimulated PMNs and fibrin was used as a measure of the interactionof these cells with fibrin-coated surfaces. About 50% of fibrin-adherentLTB4- or IL-8-stimulated PMNs formed adhesive zones that excluded thishigh molecular weight (100 kDa.) probe. As expected from studies withRh-PEG (FIG. 8), >99% of fibrin-adherent TNF- or fMLP-stimulated PMNsexcluded fluorescein-conjugated F(ab)₂ anti-fibrin from their zones ofcontact with fibrin (data not shown). Thus, LTB4- or IL-8-stimulatedPMNs adhere more closely to fibrin than unstimulated PMNs, even thoughthese chemoattractants do not promote the very close appositioncharacteristic of fMLP- or TNF-stimulated PMNs.

The Effect of Combinations of Chemoattractants on Migration of PMNsthrough Fibrin Gels

[0076] The inability of fMLP- or TNF-stimulated PMNs to migrate throughfibrin can be interpreted in at least two ways. First, fibrin blocks thecapacity of PMNs to respond to fMLP or TNF. This seems unlikely sincefMLP and TNF promote close apposition between PMNs and fibrin coatedsurfaces (FIG. 8). Second, fMLP or TNF signal PMNs to become sessilewhen they interact with fibrin. To examine the second possibility, theeffects of combinations of chemoattractants on PMN migration throughfibrin gels were monitored (FIGS. 9A and 9B). The presence of fMLP inthe bottom compartment of the inserts reduced PMN migration throughfibrin gels in response to IL-8 or LTB4 in a concentration dependentfashion. Higher concentrations of fMLP were required to effect equalinhibition of migration of LTB4-stimulated PMNs vs IL-8 stimulated PMNs(FIG. 9B). TNF had a small, reproducible, but statisticallyinsignificant inhibitory effect on the migration of PMNs in response toIL-8 and no measurable effect on PMN migration in response to LTB4 (FIG.9A). Thus, fMLP selectively reduced PMN migration through fibrin gels inresponse to IL-8 or LTB4 (FIG. 9B).

[0077] The effects of combinations of chemoattractants on formation ofclose apposition of adhesion of PMNs with fibrin were also examined.fMLP in combination with IL-8 or LTB4 induced about 50% of PMNs to formzones of adhesion that excluded 10 kDa. Rh-PEG (FIG. 8). In contrast,only 15% of PMNs stimulated with IL-8 or LTB4 alone formed close zonesof adhesion (FIG. 8). Thus, the capacity of PMNs to form close zones ofadhesion on fibrin was inversely associated with the capacity of PMNs tomigrate through fibrin gels under conditions where PMNs were stimulatedwith TNF, fMLP, IL-8 or LTB4 given alone or with fMLP in combinationwith IL-8 or LTB4.

[0078] To determine the machanism by which fibrin blocks PMN migrationin response to fMLP, the effects of anti-integrin antibodies were tested(FIGS. 10A and 10B). PMNs incubated with anti-β₁ integrin antibodies (2μg/ml) or the peptide GRGDSP (SEQUENCE ID NO. 1) (1 mg/ml), but notGRGESP (SEQUENCE ID NO. 2), migrated through fibrin gels in response tofMLP. Control experiments showed that anti-β₁ antibodies did not affectLTB4-stimulated PMN migration through fibrin. These studies show thatinteractions between PMN β₁ integrins and matrix-associated ligandsregulate PMN migration. They suggest that fMLP, but not LTB4 signalsbinding of β₁ integrins to β₁ ligands (e.g. RDG) on fibrin. Thesestudies also suggest that ligation of β₁ integrins signalsfMLP-stimulated PMN to become sessile, and that by blocking β₁ integrinswith antibodies or peptides, PMNs are able to migrate into tissue sitescontaining fibrin, from which PMN would otherwise be excluded.

Discussion Matrix Proteins Modulate Cellular Responses to Hormones,Cytokines, and Growth Factors

[0079] Matrix proteins exert profound effects on adhesion,differentiation, migration, and/or secretion of epithelial cells (16,17), endothelial cells (18), neurons (19, 20) and leukocytes (7, 9,21-27). Matrix proteins also affect the ability of many types of cellsto respond to hormones, growth factors, and cytokines (28, 29). Thefindings that some chemoattractants (e.g., fMLP, TNF, C5a), promote PMNmigration in the context of two types of extracellular matrix proteins(e.g., matrigel and collagens IV) (FIGS. 5A, 5B, 5C, 5D, 5E, 5F, 7 a,and 7B), and PMN immobilization in the context of another (e.g., fibrin)(FIGS. 1A, 1B, 2A, 5A, 5B, 5C, 5D, 5E, and 5F), are the first to showthat specific matrix proteins regulate leukocyte chemotaxis. They showthat fibrin gels, fibrin-impregnated collagen gels, andfibrin-containing plasma clots present selective barriers to themigration of fMLP- or TNF-stimulated PMNs and that chemotaxis of PMNsthrough three dimensional matrices is regulated by both the specificchemoattractant and the protein composition of the matrix with which thecells are in contact.

Matrix Proteins Regulate PMN Adhesion, Phagocytosis and Secretion

[0080] Previous studies (7, 9, 21) have shown that TNF or phorbol esterstimulated PMNs adhere to fibrinogen-coated surfaces via differentbeta-2 integrins (CD11b/CD18 vs CD11c/CD18, respectively) andLundgren-Akerlund et al. (22), and Thompson and Matsushima (23), havereported that fMLP stimulated PMNs adhere to protein coated surfaceswith different efficiencies depending on the matrix protein used to coatthese surfaces. With respect to phagocytosis, Pommier et al. (24), andWright et al., (25) showed that the interaction of fibronectin with itsβ₁ integrin activates complement receptors (CD11b/CD18) on monocytes andPMNs to phagocytose C3bi-coated particles. With respect to secretion,Monboisse et al., (26, 27) reported that the interaction of unstimulatedor chemoattractant- stimulated PMNs with collagen I-coated surfacesinduces the secretion of proteolytic enzymes and O₂ ⁻. In contrast,preincubation of PMNs with collagen IV blocks the ability of collagen Iand of fMLP to stimulate resting PMNs to secrete these products (26,27). Similarly, adhesion of TNF-stimulated PMNs to extracellular matrixproteins that express Arg-Gly-Asp motifs enhances PMN secretion (30).The findings reported here add chemotaxis to the list of leukocytefunctions modulated by their contact with matrix proteins.

Relationship Between Strength of Adhesion, Closeness of PMN Appositionto the Substrate, and PMN Migration

[0081] DiMilla at al (31), have explored the relationship betweenstrength of cell adhesion to a substrate and cell migration by followingthe spontaneous migration of human smooth muscle cells on surfaces thathad absorbed varying concentrations of fibronectin or collagen IV. Underthe conditions of their experiments, the rate of cell migration wasmaximal at an intermediate level of cell-substratum adhesiveness.Goodman et al. (32), found a similar biphasic relationship between themovement of murine skeletal myoblasts and the absorbed concentration oflaminin on the substrate.

[0082] While the strength of PMN adhesion to fibrin was not measureddirectly, the “closeness” of apposition between PMNs' matrix-adherentsurfaces and matrices containing different proteins was examined bymeasuring the permeability of zones of contact between PMNs and theunderlying matrix to macromolecular probes. “Close” apposition isdefined as the exclusion of 10 kDa Rh-PEG from zones of contact betweenthe PMNs' substrate-adherent membranes and the matrix, and “loose”apposition as permeation of 10 kDa Rh-PEG into these zones. Theseresults show that chemoattractants, such as IL-8 and LTB4, elicit“loose” apposition between PMNs and fibrin gels and promote PMNmigration through these gels. Chemoattractants, such as fMLP and TNF,that signal “close” apposition between PMNs and fibrin gels do notpromote PMN migration through these gels. This correlation was furthersupported by the findings that PMNs stimulated by any of thesechemoattractants formed loose zones of adhesion (e.g., permeable to 10kDa Rh-PEG) on collagen IV or Matrigel (FIG. 8), and migrated throughthese matrices (FIG. 7A); and that fMLP induced LTB4 or IL-8 stimulatedPMNs to form close zones of apposition to fibrin and cease migration(FIGS. 8, 9A and 9B). Thus, there is an inverse association betweenclose PMN interaction with a matrix protein and the ability of PMNs tomigrate though gels containing it. These findings suggest that “close”and “loose” apposition between PMNs and matrix proteins as defined here,are functionally equivalent to very strong and intermediate adhesionbetween cells and matrix, respectively, as defined by DiMilla et al(31).

Fibrin Degradation is Not Required for PMN Chemotaxis

[0083] The zones of close apposition formed between fMLP- orTNF-stimulated PMNs and fibrin gels are impermeant to molecules of >10kDa thereby excluding virtually all plasma protease inhibitors such asalpha₁ anti-plasmin and alpha₂ macroglobulin. Therefore, leukocyteproteases secreted into these zones function virtually uninhibited (33).In contrast, IL-8- or LTB4-stimulated PMNs adhere more loosely tofibrin-gels. Under these conditions, plasma protease inhibitors shouldhave ready access to zones of contact with the substrate and inhibit theaction of leukocyte proteases. Thus, fMLP- or TNF-stimulated PMNs mightbe expected to digest fibrin gels more efficiently than IL-8- orLTB4-stimulated PMNs. This was not observed (FIG. 6). There were nosignificant differences in the amount of radiolabel released from¹²⁵I-labeled fibrin by migrating LTB4- or IL-8-stimulated PMNs vssessile fMLP- or TNF-stimulated PMNs, even in the presence of 20% serum.These findings suggest that PMNs that migrate through fibrin gels inresponse to IL-8 and LTB4 do so by mechanisms other than proteolyzingthese gels. Lanir et al., (34) came to a similar conclusion in theirstudies of guinea pig macrophage migration through fibrin gels.

[0084] That fMLP and TNF promote PMN migration through fibrinogen-coatedfilters (FIGS. 7A and 7B), is probably related to the observations thatfMLP-stimulated PMNs efficiently degrade substrate-adherent proteinsincluding fibrinogen (33), and fibronectin (35), thereby removing theseproteins from the substrate and facilitating PMN movement.

How Do Matrix Proteins Regulate Leukocyte Chemotaxis?

[0085] The results shown in Example 1 indicate the following: Differentchemoattractants activate different subsets of PMN integrins to bind toligands on matrix proteins (7-9). The interaction of each type ofactivated PMN integrin with its cognate ligand on a matrix proteinspecifies a distinct set of cellular migratory or sessile responses.These responses may result from direct interaction of a matrix proteinwith the activated integrin or by signals sent by the activated integrinto other integrins on the same cell. There are several instances whereligation of one type of integrin by matrix proteins modulates theactivity of another type of integrin. As described above, Pommier etal., (24) and Wright et al. (25) showed that ligation of β₁ integrins byfibronectin activates the β₂ integrin CD11b/CD18, (complement receptor3) on monocytes and PMNs to phagocytose C3bi-coated particles. It waspreviously shown that ligation of α₅β₁ on platelets by fibronectinstimulates platelets to form close zones of apposition with fibrinogen(10). Hutalia et al, (36) reported that ligation of α₅β₂ integrin by RGDpeptides induces the expression of matrix metalloproteinases byfibroblasts, whereas ligation α₄β₁ integrin by intact fibronectinsuppresses matrix metalloproteinase expression.

[0086] In vivo inflammatory stimuli elicit the generation of multiplechemoattractants/cytokines. The present findings show that a hierarchyof cellular responses is generated when different combinations ofchemoattractant receptors are stimulated simultaneously. Signalsgenerated by fMLP receptors appear to override signals produced by LTB4or IL-8 receptors, thereby blocking the ability of LTB4 or IL-8 tostimulate PMN migration through fibrin gels (FIG. 8). In contrast,signals generated by TNF receptors have no effect on LTB4-stimulated PMNmigration through fibrin gels, and a very weak inhibitory effect onIL-8-stimulated PMN migration through these gels (FIG. 8).

[0087] PMN chemotaxis through three dimensional lattices composed ofextracellular matrix proteins is regulated both by signals initiated bya specific chemoattractant, and by signals generated when specific PMNreceptors interact with their cognate ligands on extracellular matrixproteins. Viewed from this perspective each of the many differentchemoattractants provides PMNs both with general instructions to crawl,and with specific instructions to become sessile when specific receptorson these cells contact their cognate ligands on matrix proteins. Thus,chemoattractants provide tissue localization instructions for PMNs. Itseems likely that chemoattractants also provide such instructions toother types of leukocytes as well.

[0088] PMNs stimulated with zymosan-activated human plasma (C5a) did notmigrate across fibrin-coated inserts but did migrate acrossmatrigel-coated inserts. Thus, C5a, like fMLP and TNF, signals PMNs tostop migrating when they contact fibrin.

Example 2

[0089] The inhibitory effects of extracellular matrix proteins onchemotaxis of leukocytes (7, 8, 9, 65) prompted the examination theeffects of tenascin on these processes. The present findings show thattenascin blocks chemotaxis of polymorphonuclear and mononuclearphagocytes across reconstituted basement membrane (Matrigel)-coatedfilters in a β₁ integrin-dependent process.

Experimental Procedures Cells

[0090] Polymorphonuclear leukocytes (PMN), were prepared as described(7) from heparinized human blood by sedimentation on Ficoll-Hypaquegradients. Contaminating red blood cells were removed by hypotoniclysis. The purity of PMN isolated by this method was >95% as determinedby Wright-Giemsa staining.

[0091] Mononuclear cells were isolated by centrifugation of heparinizedhuman blood on Ficoll-Hypaque gradients as described (65, 66). Themononuclear cell fraction was resuspended in RMPI 1640 mediumsupplemented with 10% pooled human serum or autologous serum and usedimmediately for monocyte migration studies. For some experiments,monocytes were obtained by centrifugation of whole blood or of whiteblood cells concentrated from a unit of blood (leukopak), on Nycodenzgradients as described (39). More than 90% of the nucleated cellsobtained by this Nycodenz method were monocytes, as assessed by theirability to phagocytose IgG-coated red blood cells.

[0092] Cultured monocytes were prepared by allowing 10⁷ totalmononuclear cells, suspended in 10 ml of RMPI 1640 medium supplementedwith 10% pooled human serum (1640+HS), or 10% autologous serum(1640+AS), to adhere to Falcon T-150 tissue culture Petri dishes for 2 hat 37° C. Non-adherent cells were removed by washing, leaving anadherent cell population consisting of greater than 98% monocytes asmeasured by their capacity to phagocytose IgG coated sheep red bloodcells. For migration studies, monocytes were maintained in culture for24 h in RPMI 1640+HS or AS, and detached from the dishes by gentlepipetting of 10 ml of ice cold phosphate buffered saline (without Ca²⁺or Mg²⁺) containing 1.0 mM EDTA. The cells recovered were resuspended inRPMI-1640+HS as described (65).

Protein-Coated Filters

[0093] Cell culture inserts containing polyethylene terephthalatefilters, 8-μm pore size (Becton-Dickinson), were overlaid with 0.1 ml ofMatrigel (20-25 μg protein/filter) (Collaborative Research, Bedford,Mass.), and incubated at room temperature until they dried. TheseMatrigel-coated filters were washed with phosphate buffered salinecontaining 1.0 mM Mg²⁺ and 1.0 mM Ca²⁺ (PBS). 0.1 ml of a PBS solution(pH 7.2) containing the indicated amount of purified chick braintenascin was added to some of the inserts. The filters were incubated atroom temperature until they dried. These protein-coated filters werewashed again with PBS and used within 12 h for cell migration studies.To prepare collagen I-coated inserts, filters were coated with rat tailcollagen I (ICN Biochemicals, Costa, Mesa, Calif.) (400 μg/ml in PBS),by adding 0.1 ml of this solution to an insert and incubating theinserts at room temperature for 24 h. Some of these collagen I-coatedfilters were then incubated with tenascin as described above.

Cell Migration

[0094] Monocytes: Cell culture inserts were placed in 16 mm wellscontaining 0.5 mls of RPMI-1640+HS or AS in the presence or absence of achemoattractant. 0.5 ml of RPMI-1640+HS or AS serum containing between2-10×10⁵ mononuclear phagocytes was added to the upper chamber of theinserts and the inserts were placed in a humidified CO₂ incubator at 37°C. After 24 h the medium in the lower chamber was recovered and its cellcontent assayed using either a Coulter counter or a hemocytometer. Nosignificant increase was observed in the number of monocytes thatmigrated into the lower compartment at times greater than 24 h. Cellcounts reported are the average number of cells recovered from themedium in the lower compartment of chemotaxis chambers. Over 90% of thecells in the lower chamber were identified as monocytes based on theirmorphology, their capacity to phagocytose IgG coated sheep red cells,and their staining with fluorescein-conjugated anti α,β monoclonalantibody (Oncogene Sciences, Uniondale, N.Y.). Unless otherwiseindicated, all values are the average of duplicate samples run inparallel in a representative experiment. Each experiment was repeated atleast three times with similar results.

[0095] PMN: 250 μl of PBS [supplemented with 5.5 mM glucose and 0.1%human serum albumin (HSA) (PBSG-HSA)] containing 1×10⁶ PMNs was placedin the upper compartment of each insert. 250 μl of PBSG-HSA with orwithout chemoattractant, as indicated, was added to the bottomcompartment. The inserts were incubated for about 4 h at 37° C. in ahumidified atmosphere containing 95% air/5% CO₂. No further increase inthe number of PMNs was observed in the lower compartment beyond 4 h. Themedium in the lower compartment was collected and its content of PMNdetermined using a Coulter Counter, as described in Example 1.

Tenascin

[0096] To isolate tenascin, 14-day embryonic chick brains werehomogenized in the presence of protease inhibitors and the extracts wereclarified as previously described (61). Dry CsCl was added to a finalconcentration of 0.5 g/ml and the extract was centrifuged (18 h, 45,000rpm, 20° C.) in a Beckman VAC 50 rotor. The resulting density gradientswere fractionated into five 8-ml fractions. The third and fourthfractions a rich source of relatively pure tenascin, were pooled,dialyzed versus 10 mM Tris pH 8.0, then incubated with chondroitin ABClyase (Seikagaka America), in the presence of protease inhibitors (61),to degrade contaminating proteoglycans. The sample then was lyophilized,resuspended in 4 M guanidine-HCl/0.1 M Tris (pH 7.6), and fractionatedon a 1.5×100 cm column containing Sephacryl S-500 (Pharmacia,Piscataway, N.J.), equilibrated in the same buffer. Tenascin-richfractions were pooled, dialyzed, lyophilized, resuspended in a smallvolume of guanidine buffer, and finally dialyzed extensively vs PBS.This procedure yielded large amounts of purified tenascin which migratedas characteristic 220, 200, and 190 kD polypeptides when analyzed bySDS-PAGE under reducing conditions (44). Human tenascin was obtainedfrom GIBCO-BRL, (Grand Island, N.Y.). To remove the detergent in thepreparation, the sample was run over a gel filtration column in thepresence of 4 M guanidine-HCl; the tenascin containing fractions werethen dialyzed extensively vs PBS.

Measurement of Tenascin Bound to Matrices

[0097] Chick tenascin was radiolabeled using chloramine T (67), andmixed with unlabeled tenascin at a 1:100 protein ratio. 250 μl of PBScontaining varying amounts of this mixture was added to inserts coatedwith Matrigel or collagen I. The inserts were incubated at roomtemperature for 4 h at 37° C. in a humidified 95%air/5% CO₂ atmosphereand washed with PBS. The filters were cut from the inserts with ascalpel, and assayed for 125I in a Beckman gamma counter.

Reagents

[0098] Monoclonal antibody P4C10 (anti-β chain of human beta-1integrin), was from GIBCO-BRL. Fluorescein conjugated monoclonalantibody against the β chain of human beta-2 integrins (anti-β₂-CP14F)was obtained from Oncogene Sciences (Uniondale, N.Y.). Fluoresceinconjugated anti CD11b monoclonal antibody was from AMAC Inc (Westbrook,Me.). Monoclonal anti-CD11c/CD18 (LeuM5) was from Organon-Toknika Inc.(Malvern, Pa.). F(ab)′₂ fragments of anti-tenascin antibodies wereprepared as described (46).

[0099] Chondroitin sulfate proteoglycan monomers were purified andantibodies prepared against these proteoglycan monomers as described(37). The F(ab)′₂ fragments used in the present study were prepared fromtotal anti-proteoglycan IgG. These antibodies were not further purifiedby affinity chromatography and therefore recognize both 400 kD and 250kD proteoglycan core proteins (67).

Results Tenascin Blocks Chemotaxis of Monocytes and Neutrophils, throughMatrigel-Coated Filters

[0100] About 20% of freshly isolated human monocytes, 15% of culturedmonocytes and 10% of PMNs migrated through Matrigel-coated cultureinserts in response to fMLP, LTB4, or TNF (FIGS. 11A, 11B, and 12).Monocytes began to appear in the lower compartment at 12 h, and reachedmaximum numbers by 16-24 h. PMNs began to appear in the lowercompartment by 2 h, and reached a maximum by 4 h. Fewer than 2% of addedmonocytes or PMN, migrated into the lower compartment in the absence ofa chemoattractant (FIGS. 11A, 11B, and 12).

[0101] Addition of chick brain tenascin to the Matrigel significantlyreduced monocyte and PMN migration in response to TNF, fMLP or LTB4(FIGS. 11A, 11B and 12). The presence of tenascin had no significanteffect on the limited number of monocytes that migrated in the absenceof chemoattractant (FIGS. 11A and 11B). In contrast, tenascin furtherreduced the small number of PMN that migrated across Matrigel in theabsence of chemoattractant (FIG. 12). The extent to which tenascininhibited chemoattractant-stimulated monocyte or PMN migration variedfrom 65-80% depending on the chemoattractant used (FIGS. 11A, 11B, and12). The ability of tenascin obtained from cultured human glioma cellsto effectively inhibit leukocyte migration was examined. Indeed, theaddition of 5 μg of human tenascin to the Matrigel-coated filtersreduced the number of monocytes or PMNs migrating in response to achemoattractant by at least 50% (FIGS. 13A and 13B).

[0102] The effect of chick brain tenascin on TNF-stimulated migration ofmonocytes varied with the amount of tenascin added (FIG. 14). Additionof about 0.75 μg tenascin caused half maximal inhibition ofTNF-stimulated monocyte migration (FIG. 14). To confirm that tenascinbound to the Matrigel, Matrigel-coated inserts were incubated withvarying amounts of concentrations of ¹²⁵I-labeled chick tenascin for 4 hat 37° C. The inserts then were washed with PBS and the boundradioactivity was determined. Near plateau binding of radiolabeledtenascin was obtained with the addition of 0.625-1.25 μg of chicktenascin to the Matrigel coated filters (FIG. 15). This resulted in theadsorption of about 0.2 μg of tenascin per filter. Once bound, less than1% of the ¹²⁵I-labeled tenascin eluted from the filters into either theupper or lower compartment of the chambers during a 4-6 h incubation at37° C. Thus, ˜99% of tenascin remained bound to Matrigel-coated filter.The capacity of tenascin to inhibit monocyte migration (FIG. 14) wasroughly proportional to the amount of tenascin that bound to the filter(FIG. 15), indicating that tenascin bound to the Matrigel matrix, notsoluble tenascin, blocked monocyte and PMN migration.

Tenascin Blocks Migration of Monocytes through Collagen I Gels

[0103] The effects of tenascin on monocyte migration through anotherextracellular matrix, collagen I were examined. Twelve percent of TNF-and 18% of LTB4-stimulated monocytes migrated through cell cultureinserts coated with collagen I (FIG. 16). Addition of chick braintenascin to collagen I coated inserts reduced TNF- or LTB4-stimulatedmonocyte migration by at least 60% (FIG. 16). Binding studies with¹²⁵I-labeled chick tenascin revealed that similar amounts of tenascinbound to collagen I-coated filters as to Matrigel-coated filters.

Effect of F(ab)′₂ Anti-Tenascin on the Migration of Monocytes AcrossFilters Coated with Matrigel and Tenascin

[0104] Monocytes or PMN were added to the upper compartment of insertscoated with Matrigel alone or with Matrigel and tenascin. F(ab)′₂anti-tenascin (2 μg/ml) was added to the medium in the upper compartmentand LTB4 (10⁻⁷ M) was added to the lower compartment. F(ab)′₂ fragmentsof anti-tenascin antibody had no significant effect on PMN migrationacross filters coated with Matrigel alone (FIG. 17A), but reducedmonocyte migration across this matrix by about 40% (FIG. 17B). However,F(ab)′₂ fragments of anti-tenascin antibody restored PMN chemotaxisacross filters coated with Matrigel and tenascin to about 75% of controlvalues (FIG. 17B), and increased substantially monocyte migration acrosstenascin coated Matrigel (FIG. 17A).

[0105] Because of their affinity for tenascin, proteoglycans maycontaminate some tenascin preparations (46). Therefore, the effects ofF(ab)′₂ fragments of polyclonal anti-proteoglycan on monocyte migrationacross filters coated with tenascin and Matrigel were examined. F(ab)′₂anti-proteoglycan (2-5 μg/ml) did not reverse tenascin's inhibitoryeffect on monocyte migration across filters coated with Matrigel andtenascin, and did not significantly affect monocyte chemotaxis acrossfilters coated with Matrigel alone. These studies suggest that tenascininhibits migration by interacting with monocytes and not by blockingsome matrix component required for their migration.

Antibodies that Block β₁ Integrins Reverse the Inhibitory Effects ofTenascin on Monocyte and PMN Migration through Filters Coated withMatrigel and Tenascin

[0106] Endothelial cell attachment and spreading on human tenascin hasbeen shown to be partially mediated by β₁ integrins (60). Similarly,Prieto et al. (53, 54), showed that anti-β₁ integrin antibodies blockthe adhesion of glioma and carcinoma cell lines to tenascin. Therefore,the effects of anti-β₁ antibodies on monocyte and PMN chemotaxis throughfilters coated with Matrigel alone or with Matrigel and tenascin wereexamined. Monocytes or PMNs were preincubated for 30 min at 4° C. inmedium containing 2 μg/ml of the test antibody. The suspension then wasadded to the upper compartment of the inserts.

[0107] LTB4 was added to the lower compartment and the inserts wereincubated at 37° C. for 24 h for monocytes or 4 h for PMN. A monoclonalantibody directed against β₁ integrins (P4C10) had no effect on monocytechemotaxis through filters coated with Matrigel alone but reversed theinhibitory effect of tenascin on monocyte migration throughMatrigel/tenascin coated filters by about 50% (FIGS. 17A and 17B).Control experiments showed that monoclonal antibody P4C10 also reversedtenascin's inhibitory effect on TNF-stimulated monocyte chemotaxis byabout 60%. P4C10 reversed almost completely the inhibitory effect oftenascin on LTB4- (FIG. 17B) or TNF-stimulated chemotaxis of PMN acrossfilters coated with Matrigel and tenascin.

[0108] To confirm that the effect of P4C10 was due to its interactionwith β₁ integrins, the effects of other anti-integrin antibody, A₁₁B₁₁,on chemotaxis of monocytes through filters coated with Matrigel andtenascin were examined. A₁₁B₁₁ (2 μg/ml) blocked the inhibitory effectof tenascin on monocyte chemotaxis, allowing LTB4 stimulated monocytesto migrate through Matrigel/tenascin coated filters. In contrast, anantibody (LeuM5) directed against α_(x) (p150/95), a member of the βintegrin family found on both PMNs and monocytes, did not reversetenascin's inhibitory effect on LTB4-stimulated chemotaxis of PMN ormonocytes through Matrigel/tenascin-coated filters (FIGS. 17A and 17B).As expected, monoclonal antibody IB4, directed against leukocyte β₂integrins, inhibited chemotaxis of monocytes and PMNs across filterscoated with Matrigel alone or with both Matrigel and tenascin. Thus,antibodies directed against members of the β₂ integrin family did notreverse tenascin's inhibitory effect on monocyte and PMN chemotaxis.These results indicate that blocking the interaction of monocytes orPMNs with tenascin, either by masking the tenascin on the matrix withF(ab)′₂ anti-tenascin or by blocking β₁ integrins on the cells, reversedthe inhibitory effect of tenascin on monocyte and PMN chemotaxis.

Discussion

[0109] PMNs migrate through matrices formed by, and containing, proteinsthat are “normal” constituents of basement membranes and of the groundsubstance of interstitial spaces (e.g., collagens I and IV, laminin), inresponse to all chemoattractants tested (fMLP, TNF, C5a, IL-8, LTB4)(68, 69). In contrast, whether PMN migrate through matrices composed of,or containing, fibrin depends upon the specific chemoattractant withwhich they have been stimulated. For example, fMLP, TNF and C5astimulate PMNs to adhere tightly to fibrin gels, but not to migrate intoor through them. In contrast, IL-8 and LTB4 stimulate PMNs to migrateefficiently through these gels.

[0110] The capacity of specific chemoattractants to signal cessation ofmigration when PMNs contact fibrin suggested that this might be amechanism by which these cells are excluded from some tissuecompartments, and concentrated in others. One example, however, hardlyestablishes a general principle. Therefore, other matrix proteins thatblock PMN and monocyte chemotaxis were sought.

Tenascin Inhibits PMN and Monocyte Chemotaxis through Collagen I orMatrigel Matrices

[0111] Addition of tenascin to three-dimensional matrices formed bycollagen I or Matrigel signals cessation of movement of PMNs andmonocytes in response to all three chemoattractants tested (fMLP, LTB4,TNF) (FIGS. 11-17). The capacity of tenascin to block chemotaxis of PMNsstimulated by LTB4 is of special note since LTB4 promotes PMN migrationthrough fibrin gels (25). This finding supports our contention thatleukocyte migration through extracellular matrix is regulated by boththe proteins in the matrix and the specific chemoattractant. Itdemonstrates that a chemoattractant can have entirely different effectson a single class of leukocyte, depending upon the matrix proteins withwhich the leukocyte is in contact.

One of Tenascin's Functions in Adults is to Inhibit PMN and MonocyteEntry into Specific Tissue Compartments

[0112] Tenascin is an unusual matrix protein. It is expressed widely inembryonic tissues where it regulates cell migration duringorganogenesis. Under physiological conditions in adults, tenascin isabsent from most tissues, except lymphoid tissue (40, 55), and brain(43). However, under pathological conditions, tenascin synthesis isstimulated. It is deposited in the extracellular matrix in areas ofvascular injury (57), and tumor stroma (43, 63), which are also areas offibrin deposition (59, 70-73).

[0113] It is notable that tenascin and fibrin, matrix proteins depositedin and around diseased (e.g., malignant tumors), or injured tissues(e.g., atherosclerotic lesions), or areas in which T-cells concentrate(40, 43, 55, 57, 59, 63, 70-73), and chemically modified matrix proteins(e.g., non-enzymatically glycated collagen IV [74]), all signalphagocytic leukocytes to become sessile. Dvorak et al. (70), and Singhet al. (59) have presented evidence that tumor stroma protects thetumors from host immune effector cells. Viewed from this perspective,tenascin contributes to an immuno-inhibitory effect of tumor stroma.

β₁ Integrins Play No Role in PMN or Monocyte Migration through Matrigel

[0114] Anti-β₁ integrins had no inhibitory effect on PMN or monocytechemotaxis through Matrigel alone (FIGS. 17A and 17B), while antibodiesdirected against PMN and monocyte β₂ integrins blocked PMN and monocytechemotaxis under all circumstances tested (75), including throughMatrigel (FIGS. 17A and 17B). These findings suggest that β₁ integrinsplay no role in PMN or monocyte chemotaxis through Matrigel. Thesestudies did not examine whether antibodies directed against β₂ integrinsinhibited PMN or monocyte chemotaxis through Matrigel by blocking theiradhesion to the Matrigel or by other mechanisms, such as stimulating anincrease in their cAMP content (76).

On the Mechanism(s) by which Tenascin Blocks PMN and Monocyte Chemotaxis

[0115] Chemoattractants that signal PMNs to remain sessile on fibringels, cause these cells to adhere more tightly and in greater numbers tofibrin than chemoattractants that promote PMN to migrate through fibringels. Similarly, chemoattractants stimulate monocytes to become sessileand adhere more tightly to glycated collagen IV than to native collagenIV (65, 74). In contrast, no increase in the number ofchemoattractant-stimulated PMN or monocytes that adhered totenascin-impregnated Matrigel over Matrigel alone was observed. Thus,while fibrin and glycated matrices may inhibit chemotaxis by providingligands to which chemoattractant-stimulated PMNs and monocytes bind verytightly, and in increased numbers, tenascin appears to exert itsinhibitory effect by a different mechanism.

[0116] β₁ integrins have been reported to promote adhesion of normal andtransformed cells to tenascin (53, 54, 60). F(ab)′₂ anti-tenascin, andanti-β₁ integrins reversed tenascin's inhibitory effect on both PMN andmonocyte chemotaxis (FIGS. 17A and 17B). Since β₁ integrins appear toplay no role in PMN or monocyte chemotaxis through Matrigel, the moststraightforward explanation for the capacity of F(ab)′₂ anti-tenascinand anti-β₁ integrins to block tenascin's inhibitory effect onchemotaxis, is that the interaction of β₁ integrins on PMN and monocyteswith cognate ligands on tenascin signals these cells to stop migrating.

[0117] The results of Example 1 show that β₁ integrins regulate themigration of fMLP-stimulated PMNs through fibrin and that antibodies orpeptides that block β₁ integrins allow all PMNs to migrate throughfibrin gel in response to chemoattractant that otherwise would causePMNs to stop migrating when they encounter fibrin. Further work isneeded to identify the cellular pathways via which β integrins signalPMNs and monocytes to stop moving, and to determine the physiologicalsignificance of this event. Cellular pathways and physiologicalsignificance notwithstanding, one practical consequence of thesefindings is that antibodies vs PMN and monocyte 3 integrins may betherapeutically useful by facilitating entry of these cells into tissuesand body compartments from which they otherwise are excluded.

[0118] Tenascin has domains (53), which are homologous to regions inepidermal growth factor, fibronectin, and fibrinogen. Since fibrin andtenascin block monocyte and PMN migration, tenascin's fibrinogen-liketerminal knob may play a critical role in signalling leukocytes to stopmigrating.

Example 3

[0119] The role of β₁ integrins in PMN chemotaxis through fibrin gelsformed in cell culture insers (pore size=8 μm) has been examined. 10⁶PMN were added to the upper compartment of the inserts and achemoattractant was added to the lower compartment. Previous studies(Loike, et al., J. Exp. Med. 181: 1763, 1995) showed that PMN do notmigrate through fibrin gels in response to a gradient FMLP but do soefficiently in response to a gradient of LTB4. In contrast, PMN migratethrough collagen gels in response to either FMLP or LTB4. To determinethe mechanism by which fibrin blocks PMN migration in response to FMLP,the effects of anti-integrin antibodies were tested. PMN incubated withanti-β₁ integrin antibodies (2 μg/ml) or the peptide GRGDSP (1 mg/ml),but do not GRGESP, migrated through fibrin gels in response to FMLP(FIG. 10B). Control experiments showed that anti-β₁ antibodies did notaffect LTB4-stimulated PMN migration through fibrin. Furthermore,anti-α_(v) or anti-β integrin antibodies blocked PMN migration inresponse to either FMLP or LTB4 (FIG. 10A). These studies show thatinteractions between PMN β₁ integrins and matrix-associated ligandsregulate PMN migration. They suggest that: 1) FMLP, but not LTB4,signals binding of β₁ integrins to β₁ lignads (e.g. RGD) on fibrin, 2)ligation of β₁ integrins signals FMLP-stimulated PMN to become sessile,and 3) chemoattractants signal PMN to crawl or to become sessile,depending upon the matrix proteins with which these cells are incontact.

REFERENCES

[0120] 1. Rot, A. (1991). The role of leukocyte chemotaxis ininflammation. Biochemistry of Inflammation S. W. Evans and J. T.Whicher, eds). Kluwer Academic Publishers, Lancaster, 39-54.

[0121] 2. Miller, M. D., and Krangel, M. S. (1992). Biology andbiochemistry of the chemokines: a family of chemotactic and inflammatorycytokines. Crit. Rev. Immunol. 12:17-46.

[0122] 3. Snyderman, R., and Uhing, R. J. (1992). Chemoattractantstimulus-response coupling. Inflammation: basic principles and clinicalcorrelates. J. I. Gallin, I. M. Goldstein, and R. Snyderman, eds. RavenPress, New York. 421-439.

[0123] 4. Kishimoto, T. K., and Anderson, D. C. (1992). The role ofintegrins in inflammation. Inflammation: basic principles and clinicalcorrelates. J. I. Gallin, I. M. Goldstein, and R. Snyderman, eds. RavenPress, New York. 353-406.

[0124] 5. Lasky, L. A., and Rosen S. D. (1992). The selectins.Carbohydrate binding adhesion molecules of the immune system.Inflammation: basic principles and clinical correlates. J. I. Gallin, I.M. Goldstein, and R. Snyderman, eds. Raven Press, New York. 407-420.

[0125] 6. Downey, G. P. (1994). Mechanisms of leukocyte motility andchemotaxis. Current Opinion in Immunol. 6:113-124.

[0126] 7. Loike, J. D., Sodeik, B., Cao, L., Leucona, S., Weitz, J. I.,Detmers, P. A., Wright, S. D., and Silverstein, S. C. (1991). CD11c/CD18on Neutrophils recognizes a domain at the N terminus of the Aα offibrinogen. Proc. Natl. Acad. Sci. 88:1044-1048.

[0127] 8. Loike, J. D., Silverstein, R., Wright, S. D., Weitz, J. I.,and Silverstein, S. C. (1992). The role of protected extracellularcompartments in interactions between leukocytes, and platelets andfibrin/fibrinogen matrices. Plasminogen activation in fibrinolysis, intissue remodeling, and in development. P. Brakman, and C. Kluft.Editors. Ann. N.Y. Acad. Sci. 667:163-172.

[0128] 9. Wright, S. D., Weitz, J. I., Huang, A. J., Levin, J. M.,Silverstein, S. C., and Loike, J. D. (1988). Complement receptor typethree (CD11b/CD18) of human polymorphonuclear leukocytes recognizesfibrinogen. Proc. Natl. Acad. Sci. 85, 7734-7738.

[0129] 10. Loike, J. D., Silverstein, R., Cao, L., Solomon, L., Weitz,J. I., Haber, E., Matsueda, G. R., Bernatowicz, M. S., and Silverstein,S. C. (1993). Activated platelets form protected zones of adhesion withfibrinogen and fibronectin-coated surfaces. J. Cell Biol. 121:945-955.

[0130] 11. Smith, W. B., Gamble, J. R., Clark-Lewis, I., and Vadas, M.A. (1993). Chemotactic desensitization of neutrophils demonstrates IL 8dependent and IL-8 independent mechanisms of transmigration throughcytokine activated endothelium. Immunology 78:491-497.

[0131] 12. Zigmond, S. H., and Hirsch, J. G. (1973). Leukocytelocomotion and chemotaxis. New methods for evaluation, and demonstrationof a cell derived chemotactic factor. J. Exp. Med. 137: 387-410.

[0132] 13. Harvath L., and Leonard, E. J. (1982). Two neutrophilpopulations in human blood with different chemotactic activities:separation and chemoattractant binding. Infect. Immunol. 36:443-449.

[0133] 14. Furie M. B., Naperstek, B. L., and Silverstein, S. C. (1987).Migration of neutrophils across monolayers of cultured microvascularendothelial cells. An in vitro model of leucocyte extravasation. J.Cell. Sci. 88:161-175.

[0134] 15. Weston, S. A. and Parish, C. R. (1990). New fluorescent dyesfor lymphocyte migration studies. Analysis by flow cytometry andfluorescence microscopy. J. Immunol. Meth. 133:87-97.

[0135] 16. Howlett, A. R., Bissell, M. J. (1993). The influence oftissue microenvironment (stroma and extracellular matrix) on thedevelopment and function of mammary eptithelium. Epithelila Cell Biol.2:79-89.

[0136] 17. Klemke, R. L., Yebra, M., Bayna, E. M., and Cheresh, D. A.(1994). αvβ5 directed cell mobility but not adhesion on vitronectin. J.Cell Biol. 127:859-866.

[0137] 18. Milici, A. J., Furie, M. B., and Carly, W. W. (1985). Theformation of fenestration and channels by capillary endothelium invitro. Proc. Natl. Acad. Sci. 82:6181-6185.

[0138] 19. Calof A. L., and Lander, A. D. (1991). Relationship betweenneuronal migration and cell-substratum adhesion: laminin and merosinpromote olfactory neuronal migration but are anti-adhesive. J. CellBiol. 115:779-794.

[0139] 20. Bronner-Fraser, M. (1994). Neural crest cell formation andmigration in the developing embryo. FASEB J. 8:699-706.

[0140] 21. Detmers, P. A., Lo, S. K., Olsen-Egbert, E., Walz, A.,Baggiolini, M., and Cohn, Z. A. (1990). Neutrophil-activating protein1/interleukin 8 stimulates the binding activity of the leukocyteadhesion receptor CD11b/CD18 on human neutrophils. J. Exp. Med. 171,1155-1162.

[0141] 22. Lundgren-Akerlund, E., Berger, E., and Arfors, K. E. (1992).Effect of divalent cations on adhesion of PMNs to matrix molecules invitro. J. Leuk. Biol. 51: 603-608.

[0142] 23. Thompson, H. L., and Matsushima, K. (1992). Humanpolymorphonuclear leucocytes stimulated by TNF-α show increasedadherence to extracellular matrix proteins which is mediated via theCD11b/18 complex. Clin. Exp. Immunol. 90:280-285.

[0143] 24. Pommier C. G., Inada, S., Fries, L. F.. Takahashi, T., Frank,M. M., and Brown, E. J. (1983). Plasma fibronectin enhances phagocytosisof opsonized particles by human peripheral blood monocytes. J. Exp. Med.157:1844-1854.

[0144] 25. Wright, S. D., Licht, M. R., Craigmyle, L. S., andSilverstein, S. C. (1985). Communication between receptors for differentligands on a single cell: ligation of fibronectin receptors induces areversible alteration in the function of complement receptors oncultured human monocytes. J. Cell Biol. 99:336-339.

[0145] 26. Monboisse, J-C., Bellon, G., Randoux, A., Duffer, J., andBorel, J. P. (1990). Biochem. J. 270:459-462.

[0146] 27. Monboisse, J-C., Garnotel, R., Bellon, G., Ohno, N., Perreau,C., Borel J. P., and Kefalides. (1994). The α3 chain of type IV collagenprevents activation of human polymorphonuclear leukocytes. J. Biol.Chem. 269: 25475-25482.

[0147] 28. Nicosia, R. F. and Tuszynski, G. P. (1994). Matrix-boundthrombospondin promotes angiogenesis in vitro. J. Cell Biol.124:183-193.

[0148] 29. Ohno, K., and Maier., P. (1994). Cultured rat hepatocytesadapt their cellular glycolytic activity and adenylate energy status totissue oxygen tension: influences of extracellular matrix components,insulin and glucagon. J. Cell. Physiol. 160:358-366.

[0149] 30. Fuortes, M., Jen, W-W., and Nathan, C. (1993).Adhesion-dependent Protein Tyrosine Phosphorylation in NeutrophilsTreated with Tumor Necrosis Factor. J. Cell Biology 120: 777-784.

[0150] 31. DiMilla, P. A., Stone, J. A., Quinn, J. A., Albelda, S. M.,and Lauffenburger, D. A. (1993). Maximal migration of human smoothmuscle cells on fibronectin and type IV collagen occurs at anintermediate attachment strength. J. Cell Biol. 122:729-737.

[0151] 32. Goodman, S. L., Risse, G., and von der Mark, K. (1989). TheE8 subfragment of laminin promotes locomotion of myoblasts overextracellular matrix. J. Cell Biol. 109:799-809.

[0152] 33. Weitz, J. I., Huang, A. J., Landman, S. L., Nicholson, S. C.,and Silverstein, S. C. (1987). Elastase-mediated fibrinogenolysis bychemoattractant-stimulated neutrophils occurs in the presence ofphysiological concentrations of antiproteinases. J. Exp. Med.166:1836-1850.

[0153] 34. Lanir, N., Ciano, P. S., Van De Water, L., McDonagh, J.,Dvorak, A. N., and Dvorak, H. P. (1988). Macrophage migration in fibringel matrices. II. Effects of clotting factor XIII, fibronectin, andglycosaminoglycan content on cell migration. J. Immunol. 140:2340-2349.

[0154] 35. Campbell, E. J., Senior, R. M., McDonald, J. A., and Cox, D.L. (1982). Proteolysis by neutrophils. Relative importance ofcell-substract contact and oxidative inactivation of proteinaseinhibitors in vitro. J. Clin. Invest. 70:845-852.

[0155] 36. Huhtala, P., Humpries, M., McCarthy, J., Werb Z., and Damsky,C. (1994). The RGD and CS-1 containing cell binding regions offibronectin signal opposing effects on metalloproteinase expression viaα5β1. Mol. Biol. Cell 5:64a.

[0156] 37. Balsamo J, Ernst H, Zanin M K B, Hoffman S, Lilien J. (1995).The interaction of the retina cell surfaceN-acetylgalactosaminylphosphotransferase with an endogenous proteoglycanligand results in inhibition of cadherin-mediated adhesion, J. CellBiol. 129:1391-1401.

[0157] 38. Bourdon, M. A., Ruoslahti, E. (1989). Tenascin mediates cellattachment through an RGD-dependent receptor. J. Cell Biology108:1149-1155.

[0158] 39. Boyum, A., Lovhaug, D., Tresland, L., Nordlie, E. M. (1991).Separation of leucocytes: improved cell purity by fine adjustments ofgradient medium density and osmolality. Scand. J. Immunol. 34:697-712.

[0159] 40. Chilosi, M., Lestani, M., Benedetti, A., Montagna, L.,Pedron, S., Scarpa, A., Menestrina, F., Hirohashi, S., Pizzolo, G., andSemenzato, G. (1993). Constitutive expression of tenascin in T-dependentzones of human lymphoid tissues. Am. J. of Path. 143:1348-55.

[0160] 41. Chiquet-Ehrismann, R. (1993). Tenascin and otheradhesion-modulating proteins in cancer. [Review] Sem. in Cancer Biol.4:301-10.

[0161] 42. Chuong, C. M., and Chen, H. M. (1991). Enhanced expression ofneural cell adhesion molecules and tenascin (tenascin) during woundhealing. Am. J. Pathol. 138:427-440.

[0162] 43. Erickson, H. P. and Bourdon, M. A. (1989). Tenascin: anextracellular matrix protein prominent in specialized embryonic tissuesand tumors. Ann.Rev. Cell. Biol. 5:71-92.

[0163] 44. Grumet, M., Hoffman, S. Crossin, K. L., and Edelman, G. M.(1985). Tenascin, an extracellular matrix protein of neural andnon-neural tissues that mediates glial-neuron interaction. Proc. Natl.Aca. Sci. 82:8075-9079.

[0164] 45. Herlyn, M., Graeven, U., Speicher, D., Sela, B. A.,Bennicelli, J. L., Kath, R., Guerry, D. (1991). Characterization oftenascin secreted by human melanoma cells. Cancer Res. 51:4853-8.

[0165] 46. Hoffman, S., Crossin, K. L., and Edelman, G. M. (1988).Molecular forms, binding functions, and developmental expressionpatterns of cytotactin and cytotactin-binding proteoglycans, aninteractive pair of extracellular matrix molecules. J. Cell Biol.106:519-532.

[0166] 47. Hoffman, S. Dutton, S. L., Ernst, H., Boackle, M. K.,Everman, D., Tourkin, A., and Loike, J. D. (1994). Functionalcharacterization of anti-adhesion molecules. Perspectives in DevelopmentNeurbiology 2:101-110.

[0167] 48. Joshi, P., Chung, C. Y., Aukhil, I., and Erickson, H. P.(1993). Endothelial cells adhere to the RGD domain and thefibrinogen-like terminal knob of tenascin. J. Cell Sci. 106:389-400.

[0168] 49. Juhasz, I., Murphy, G. F., Yan, H. C., Herlyn, M., Albelda,S.-M. (1993). Regulation of extracellular matrix proteins and integrincell substratum adhesion receptors on epithelium during cutaneous humanwound healing in vivo. Am. J. of Path. 143:1458-69.

[0169] 50. Mackie., E. J., Halfter, W., and Liverani, D. (1988). J. CellBiol. 107:2757-2767.

[0170] 51. Mackie, E. J., Chiquet-Ehrismann, R., Pearson, C. A.,Inaguma, Y. M., Taya, K., Kawarada, Y., and Sakakura, T. (1987). Proc.Natl. Acad. Sci. 84:4621-4625.

[0171] 52. Pesheva, P., Probstmeier, R., Skubitz, A. P., McCarthy, J.B., Furcht, L. T. and Schachner, M. (1994). Tenascin-R J1 160/180inhibits fibronectin-mediated cell adhesion--functional relatedness totenascin-C. J. Cell Sci. 107:2323-33.

[0172] 53. Prieto, A. L., Andersson-Fisone, C. and Crossin, K. L.(1992). Characterization of multiple adhesive and counteradhesivedomains in the extracellular matrix protein cytotactin. J. Cell Bio.119:663-78.

[0173] 54. Prieto, A. L., Edelman, G. M., and Crossin, K. L. (1993).Multiple integrins mediate cell attachment to cytotactin/tenascin. Proc.Natl. Acad. Sci. USA 90:10154-8.

[0174] 55. Ruegg, C. R., Chiquet-Ehrismann, R., and Alkan, S. S. (1989).Tenascin, an extracellular matrix protein, exerts immunomodulatroyactivities. Proc. Natl. Acad. Sci. 86:7637-7441.

[0175] 56. Sakai, T., Kawakatsu, H., Hirota, N., Yokoyama, T., Sakakura,T., and Saito, M. (1993). Specific expression of tenascin in humancolonic neoplasms. British Journal of Cancer 67:1058-64.

[0176] 57. Sharifi, B. G., D. W. Lafleur, S. M. Schwartz, J. S.Forrester, J. A., Fagin. (1995). Expression of tenascin isoforms areselectively up-regulated following aortic balloon injury. The FASEBJournal 9: a611.

[0177] 58. Shoji, T., Kamiya, T., Tsubura, A., Hatano, T., Sakakura, T.,Yamamoto, M., Morii, S. (1992). Immunohistochemical staining patterns oftenascin in invasive breast carcinomas. Virchows. Arch. A. Pathol. Anat.Histopathol. 421:53-6.

[0178] 59. Singh, S., Ross, S. R., Acena, M., Rowly, D. A., andSchreiber, H. (1992). Stroma is critical for preventing or permittingimmunological destruction of antigenic cancer cells. J. Exp. Med.175:139-146.

[0179] 60. Sriramarao, P., Mendler, M., and Bourdon, M. A. (1993).Endothelial cell attachment and spreading on human tenascin is mediatedby alpha 2 beta 1 and alpha v beta 3 integrins. J. Cell Sci.105:1001-12.

[0180] 61. Tourkin A., Anderson T., LeRoy E C, Hoffman S. (1993).Eosinophil adhesion and maturation is modulated by laminin. CellAdhesion and Communication 1:161-176.

[0181] 62. Wehrle-Haller, B. and Chiquet, M. (1993). Dual function oftenascin: simultaneous promotion of neurite growth and inhibition ofglial migration. J. Cell Sci. 106:597-610.

[0182] 63. Hiraiwa, N., Kida, H., Sakakura, T., and Kusakabe, M. (1993).Induction of tenascin in cancer cells by interactions with embryonicmesenchyme-mediated by a diffusible factor. J. Cell. Sci. 104:289.

[0183] 64. Koukouus, G. K., Gould, V. E., Bhattacharyya, A., Gould, J.E., Howeedy, A. A., and Virtanen, I. (1991). Tenascin in normal,reactive, hyperplastic and neoplastic tissue: biological andpathological implications. Hum. Pathol. 22:636.

[0184] 65. El Khoury, J., Loike, J., Cao, L., Thomas, C., andSilverstein, S. C. (1994). Macrophages adhere to glucose-modifiedbasement membrane collagen via their scavenger receptors. J. Biol. Chem.269:10197-10200.

[0185] 66. Loike, J. D., Somes, M., and Silverstein, S. C. (1986).Creatine uptake, metabolism, and efflux in human monocytes andmacrophages. Am. J. Physiol. 251:C128.

[0186] 67. Friedlander, D. R., Hoffman, S., and Edelman, G. M. (1988).Functional mapping of cytotactin: proteolytic fragments active incell-substrate adhesion. J. Cell Biol. 107:2329.

[0187] 68. Lundgren-Akerlund, E., Olofsson, A. M., Bergerand, E., andArfors, K. E. (1993). CD11b/CD18-dependent polymorphonuclear leucocyteinteraction with matrix proteins in adhesion and migration. Scan. J.Immunol. 37:569.

[0188] 69. Islam, L. N., McKay, I. C., and Wilkinson, P. C. (1985). Theuse of collagen or fibrin gels for the assay of human neutrophilchemotaxis. J. Immunol. Meth. 85:137.

[0189] 70. Dvorak, H. F. (1986). Tumors: Wounds that do not heal.Similarities between tumor stroma generation and wound healing. N. Engl.J. Med. 315:1650-1659.

[0190] 71. Willhelm, O., Hafter, R., Coppenrath, E., Pflanz, M.,Schmitt, M., Babic, R., Linke, R., Gossner, W., and Graeff, H. (1988).Fibrin-fibronectin compounds in Human ovarian tumor ascites and theirpossible relation to he tumor stroma. Can. Res. 48:3507.

[0191] 72. Strickland D. K., Kounnas, M. Z., and Argraves, W. S. (1995).LDL receptor-related protein: a multiligand receptor for lipoprotein andproteinase catabolism. [Review] FASEB J. 9:890.

[0192] 73. Costantini, V., Zacharski, L. R., Memoli, V. A., Kisiel, W.,Kudryk, B. J., Rousseau, S. M., and Stump. D. C. (1992). TI—Fibrinogendeposition and macrophage-associated fibrin formation in malignant andnonmalignant lymphoid tissue. J. Lab. & Clin. Med. 119:124.

[0193] 74. Schmidt A. M., Yan, S. D., Brett, J., Mora, R., Nowygrod, R.,and Stern, D. (1993). Regulation of human mononuclear phagocytemigration by cell surface-binding proteins for advanced glycation endproducts. J. Clin. Invest. 91:2155.

[0194] 75. Gao, J. X., Wilkins, J., and Issekutz, A. C. (1995).Migration of human polymorphonuclear leukocytes through a synovialfibroblast barrier is mediated by both beta 2 (CD11/CD18) integrins andthe beta 1 (CD29) integrins VLA-5 and VLA-6. Cell. Immunol. 163:178.

[0195] 76. Gresham, H. D., Graham, I. L., Anderson, D. C., and Brown, E.J. (1991). Leukocyte adhesion-deficient neutrophils fail to amplifyphagocytic function in response to stimulation. Evidence forCD11b/CD18-dependent and -independent mechanisms of phagocytosis. J.Clin. Invest. 88:588.

1 2 6 amino acids amino acid single linear peptide NO NO 1 Gly Arg GlyAsp Ser Pro 1 5 6 amino acids amino acid single linear peptide NO NO 2Gly Arg Gly Glu Ser Pro 1 5

What is claimed is:
 1. A method of treating an infection caused bybacterial cells located on a surface of a foreign body over and aroundwhich fibrin has been deposited, the foreign body being present in asubject, which comprises administering to the subject an agent capableof inhibiting signalling mediated by a β₁ integrin cell surface receptorof leukocyte cells in an amount effective to enhance the migration ofleukocyte cells into or through the fibrin so as to permit the leukocytecells to reach and kill the bacterial cells and thereby treat theinfection.
 2. The method of claim 1, wherein the foreign body is aprosthetic device.
 3. The method of claim 1, wherein the foreign body isa catheter.
 4. The method of claim 1, wherein the foreign body is asuture.
 5. The method of claim 1, wherein the subject is a mammal. 6.The method of claim 5, wherein the mammal is a human.
 7. The method ofclaim 1, wherein the leukocyte cells are polymorphonuclear leukocytecells.
 8. The method of claim 7, wherein the polymorphonuclear leukocytecells are neutrophils.
 9. The method of claim 7, wherein thepolymorphonuclear leukocyte cells are basophils.
 10. The method of claim7, wherein the polymorphonuclear leukocyte cells are eosinophils. 11.The method of claim 1, wherein the leukocyte cells are monocytes. 12.The method of claim 1, wherein the leukocyte cells are macrophages. 13.The method of claim 1, wherein the agent is a peptide.
 14. The method ofclaim 13, wherein the peptide contains a β₁ integrin-binding domain. 15.The method of claim 14, wherein the peptide comprises GRGDSP.
 16. Themethod of claim 1, wherein the agent is an antibody or a fragmentthereof that specifically binds to the β₁ integrin cell surface receptorof leukocyte cells.
 17. A method of preventing a chronic infection fromoccurring due to the presence of bacterial cells on a surface of aforeign body in a subject, which comprises coating the foreign bodybefore placing it in the subject with a fibrinolytic agent capable ofpreventing the accumulation of fibrin on the surface of the foreign bodyso as to permit leukocyte cells to reach and kill any bacterial cellspresent on the surface of the foreign body and thereby prevent thechronic infection.
 18. The method of claim 17, wherein the foreign bodyis a prosthetic device.
 19. The method of claim 17, wherein the foreignbody is a catheter.
 20. The method of claim 17, wherein the foreign bodyis a suture.
 21. The method of claim 17, wherein the subject is amammal.
 22. The method of claim 21, wherein the mammal is a human. 23.The method of claim 17, wherein the fibrinolytic agent is a plasminogenactivator.
 24. The method of claim 23, wherein the plasminogen activatoris urokinase.
 25. The method of claim 23, wherein the plasminogenactivator is streptokinase.
 26. The method of claim 23, wherein theplasminogen activator is tissue plasminogen activator.
 27. A method oftreating a malignant tumor comprising of malignant tumor cells over andaround which tenascin has been deposited, the malignant tumor beingpresent in a subject, which comprises administering to the subject anagent capable of inhibiting signalling mediated by a β₁ integrin cellsurface receptor of leukocyte cells in an amount effective to enhancethe migration of leukocyte cells through the tenascin so as to permitthe leukocyte cells to reach and kill the malignant tumor cells andthereby treat the malignant tumor.
 28. The method of claim 27, whereinthe subject is a mammal.
 29. The method of claim 28, wherein the mammalis a human.
 30. The method of claim 27, wherein the leukocyte cells arepolymorphonuclear leukocyte cells.
 31. The method of claim 30, whereinthe polymorphonuclear leukocyte cells are neutrophils.
 32. The method ofclaim 30, wherein the polymorphonuclear leukocyte cells are basophils.33. The method of claim 30, wherein the polymorphonuclear leukocytecells are eosinophils.
 34. The method of claim 27, wherein the leukocytecells are monocytes.
 35. The method of claim 27, wherein the leukocytecells are macrophages.
 36. The method of claim 27, wherein the leukocytecells are lymphocyte cells.
 37. The method of claim 36, wherein thelymphocyte cells are NK cells.
 38. The method of claim 36, wherein thelymphocyte cells are Killer cells.
 39. The method of claim 27, whereinthe agent is a peptide.
 40. The method of claim 39, wherein the peptidecontains a β₁ integrin-binding domain.
 41. The method of claim 40,wherein the peptide comprises GRGDSP.
 42. The method of claim 27,wherein the agent is an antibody or a fragment thereof that specificallybinds to the β₁ integrin cell surface receptor of leukocyte cells.
 43. Amethod of treating a chronic inflammation in a subject caused by anincrease in the number of leukocyte cells present at the site of thechronic inflammation which comprises administering to the subject anagent capable of stimulating signalling mediated by a β₁ integrin cellsurface receptor of leukocyte cells in an amount effective to inhibitthe migration of leukocyte cells toward the site of the chronicinflammation so as to reduce the number of leukocyte cells present atthe site and thereby treat the chronic inflammation.
 44. The method ofclaim 43, wherein the subject is a mammal.
 45. The method of claim 44,wherein the mammal is a human.
 46. The method of claim 43, wherein theleukocyte cells are polymorphonuclear leukocyte cells.
 47. The method ofclaim 46, wherein the polymorphonuclear leukocyte cells are neutrophils.48. The method of claim 46, wherein the polymorphonuclear leukocytecells are basophils.
 49. The method of claim 46, wherein thepolymorphonuclear leukocyte cells are eosinophils.
 50. The method ofclaim 43, wherein the leukocyte cells are monocytes.
 51. The method ofclaim 43, wherein the leukocyte cells are macrophages.
 52. The method ofclaim 43, wherein the agent is a peptide.
 53. The method of claim 52,wherein the peptide contains a β₁ integrin-binding domain.
 54. Themethod of claim 53, wherein the peptide comprises GRGDSP.